Myostatin is an important regulator of body composition. Inhibition of myostatin expression in vertebrates increases muscle mass and decreases fat mass. These observations have formed the rationale for the applications of myostatin antagonists for HIV-associated weight loss and visceral obesity, and sarcopenia of aging. However, the mechanisms by which myostatin regulates muscle and fat mass are poorly understood. We will test the hypothesis that myostatin inhibits the differentiation of adult, multipotent mesenchymal progenitor cells into myogenic lineage and promotes their differentiation into adipogenic lineage. We hypothesize that myostatin regulates the differentiation of mesenchymal multipotent cells by activating SMAD pathway and cross-communicating the signal to Wnt pathway through 2 catenin and LEF/TCF-4. We will use both in vivo and in vitro approaches and employ two validated models of mesenchymal, stem cell differentiation, namely, mesenchymal multipotent C3H10T1/2 cells, and marrow-derived human mesenchymal stem cells (hMSCs). We will determine myostatin effects on myogenic and adipogenic differentiation of hMSCs, and assess whether these effects are blocked by anti-myostatin antibody. Specific Aim 2 will evaluate whether Smad2 and Smad3 act as substrates for myostatin-induced phosphorylation, form complexes with Smad4, which interacts with 2 catenin either directly or through p300. Specific Aim 3 will determine the effects of myostratin and anti-myostatin antibody on 2 catenin translocation into nucleus, a hallmark of Wnt activation. We will evaluate whether inhibition of 2 catenin by siRNA blocks myostatin effects on Wnt target genes. We will determine whether myostatin down regulates TCF4 transcription and whether inhibition of TCF4 by dominant negative cDNA blocks myostatin effects on hMSC differentiation. Specific Aim 4 will elucidate changes in intramuscular myostatin expression and signaling associated with SIV-infection and weight loss in a simian model of SIV-associated wasting. We will conduct a randomized controlled trial to determine the effects of a myostatin inhibitor on lean body mass and body weight in SIV-infected macaques experiencing weight loss. These studies will provide unique insights into the mechanisms of myostatin action and unveil novel targets for the discovery of function promoting anabolic therapies. The proposed investigations also will further our understanding of the mechanisms that regulate mesenchymal stem cell differentiation postnatally. PUBLIC HEALTH RELEVANCE: Myostatin is being explored as an attractive target for the discovery of function promoting anabolic therapies for conditions associated with loss of muscle mass and physical function, such as HIV-associated weight loss and wasting, and aging. This project aims to elucidate the mechanisms by which myostatin regulates skeletal muscle and fat mass. We will test the hypothesis that myostatin regulates mesenchymal stem cell differentiation through activation of the TGF-beta/SMAD pathway and cross-communication of the SMAD signal to the Wnt/beta-catenin/TCF-4 pathway and thereby modulating myogenic and adipogenic differentiation.